Mammalian animal compositon

ABSTRACT

The present invention relates to the use of probiotic microorganism in the manufacture of a composition for the prevention or reduction of gastrointestinal  Camplylobacter  infection in a mammalian animal. It also relates to a method for the prevention or reduction of gastrointestinal  Campylobacter  infection in a mammalian animal, the method comprising administering to said animal, a probiotic microorganism. The invention also relates to a probiotic microorganism, for use in preventing or reducing gastrointestinal  Campylobacter  infection in a mammalian animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage Application of InternationalApplication PCT/GB03/02469 filed Jun. 6, 2003, which claims priority toGreat Britain Application No. 0212975.7 filed Jun. 6, 2002.

TECHNICAL FIELD

The present invention relates to the use of a probiotic microorganism inthe manufacture of a composition for the prevention or reduction ofgastrointestinal Campylobacter infection in a mammalian animal. It alsorelates to a method for the prevention or reduction of gastrointestinalCampylobacter infection in a mammalian animal, the method comprisingadministering to said animal, a probiotic microorganism. The inventionalso relates to a probiotic microorganism, for use in preventing orreducing gastrointestinal Campylobacter infection in a mammalian animal.

BACKGROUND OF THE INVENTION

Companion animals, particularly dogs and cats, are significant vectorsof non-food borne gastrointestinal infection. Decreasing the risk ofinfection of these animals, and the ability to reduce infection when itdoes occur plays an important role in reducing zoonotic risk. Zoonoticrisk is the risk of transfer of infection from one species to another.Clearly, this includes the risk of transfer of infection from companionanimals to humans.

In dogs and cats, Campylobacter and E. coli are the predominantgastrointestinal pathogens, causing both clinical and non-clinicalinfections.

In dogs and cats, fecal shedding of Campylobacter occurs in animalswhich are infected, whether clinical symptoms are shown or not.

Campylobacter is a most common zoonoses, as well as being a common causeof gastroenteritis in humans. It is estimated that 5% of all human C.jejuni-induced enteritis result from exposure to infected dogs or cats.

In view of the zoonotic risk of Campylobacter infection from companionanimals to humans, it is recommended that control measures that shouldbe considered, which include restricting contact of children withpuppies which may be infected, pets which may be infected be kept awayfrom food preparation areas, affected animals should be kept apart fromhealthy ones and thorough disinfecting of bedding, food bowls etc shouldbe carried out.

As mentioned above, Campylobacter infection in cats and dogs may or maynot result in clinical symptoms. Thus it is difficult to know whetherany animal, at any time, is infected or not. A 3 to 7 day incubationperiod is found in dogs and cats, which may be followed by a diarrheathat ranges from mild to transient to mucus laden bloody diarrhea.However, since diarrhea is symptomatic of an enormous number ofproblems, including a range of infections, dietary problems (rapidchange, over eating, scavenging, food tolerance, food hypersensitivity),neoplasia, inflammatory bowel disease, pancreatitis, metabolic disease,systemic disease, and drug reactions, the noting of diarrhea in itselfcannot be used to diagnose Campylobacter infection.

BRIEF SUMMARY OF THE INVENTION

Accordingly, it would be of benefit to provide means to reduce orprevent Campylobacter infection in the gastrointestinal tract,particularly of companion animals. A benefit is to reduce or preventCampylobacter infection, without the need for a formal diagnosis ofCampylobacter infection. A benefit of reducing or preventingCampylobacter infection in mammalian animals results in a reduction orprevention of shedding of Campylobacter in feces and thus reduces orprevents the zoonotic risk, particularly to humans.

Accordingly, the present invention provides the use of a probioticmicroorganism in the manufacture of a composition for the prevention orreduction of gastrointestinal Campylobacter infection in a mammaliananimal.

A probiotic microorganism is one which can help to promote a healthyintestinal tract. Probiotic microorganisms beneficially affect a host byimproving the microbial balance.

The prevention or reduction of gastrointestinal Campylobacter infectionresults not only in a reduced presence of Campylobacter in the GI tract,but also, and importantly, reduces or prevents shedding of Campylobacterin feces. Reduction of the shedding of Campylobacter in feces is asignificant factor in reducing or preventing the transfer ofCampylobacter infection from animal to animal, including from companionanimal to humans.

The probiotic microorganism may be any which is known, including one ormore from the following:—

Lactobacillus (such as murinus, ruminus, rhamnosis, acidophilus, reuterior mucosae), Bifidobacterium, Bacterioides, Aostridium, Fusobacterium,Melissococcus, Propionibacterium, Streptococcus, Enterococcus,Lactococcus, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus,Micrococcus, Leuconostoc, Weisella, Aerococcus, Oenococcus andEubacterium.

Typically, the Campylobacter infection will be Campylobacter jejuni.This is the most significant strain in humans which causesgastroenteritis. The Campylobacter infection may be any other, includingCampylobacter coli, C. upsaliensis, C. lari, C. fetus, C. rectus and/orC. hyointestinalis.

The mammalian animal according to the present invention may be any.Preferably, the mammalian animal is a companion animal, such as thedomestic dog or the domestic cat. In the present invention, the termsdomestic dog and domestic cat mean dogs and cats, in particular Felisdomesticus and Canis domesticus. The present invention also applies tohumans.

The composition for the prevention or reduction of gastrointestinalCampylobacter infection may be any composition which a mammalian animalmay take. Preferably it is a composition which any mammalian animal mayconsume in its diet. Thus, the invention covers standard food productsas well as food snacks. The composition may comprise a cereal product orconfectionery, such as snack bars, biscuits and sweet products,including candy and chocolate.

When the mammalian animal is a companion animal (a pet animal) thecomposition may encompass any product which a pet may consume, inparticular in its diet. The composition is preferably a dry pet food.Such dry pet foods include dry kibbles comprising a cooked starchsource.

The foodstuff may be a cooked product. It may incorporate meat or animalderived materials (such as beef, chicken, turkey, lamb, blood plasma,marrowbone etc or two or more thereof). The composition mayalternatively be meat-free (preferably including a meat substitute suchas soya, maize gluten or a soya product). The composition may containadditional protein sources such as soya protein concentrate, milkproteins, gluten etc. The composition may contain a starch source suchas one or more grains (e.g. wheat, corn, rice, oats, barley etc) or maybe starch-free. A typical dry commercial dog and cat food contains about30% crude protein, about 10-20% fat and the remainder beingcarbohydrate, including dietary fiber and ash. A typical wet or moistproduct contains (on a dry matter basis) about 40% fat, 50% protein andthe remainder being fiber and ash. The present invention is particularlyrelevant for a composition as hereindescribed which is sold as a diet,foodstuff or supplement for a cat or dog.

Further, the composition may be a foodstuff in the form of one or moreof a cereal product, energy bar, breakfast cereal, confectionery,medicament, food supplement or a drink. The supplement may be in theform of a dried powder, tablet, capsule, liquid or gel.

The probiotic microorganism may be in any form, for example in apowdered dry form or in spore form (for the microorganisms which formspores). The probiotic may be encapsulated in order to protect it frommoisture. In addition, the probiotic microorganism may have undergoneprocessing in order for it to increase its survival in any processing.Accordingly, the microorganism may be coated or encapsulated in apolysaccharide, fat, starch, protein or in a sugar matrix. The probioticmicroorganism may be in a coating (outer or a layer), or a filling, orit may be admixed throughout the composition.

It may be preferable to avoid the probiotic being in contact with flouras flour contains enzymes which may adversely affect the viability ofthe probiotic. Standard encapsulation techniques known in the art can beused, and for example, as discussed in U.S. Pat. No. 6,190,591 (which isincorporated by reference herein).

The composition according to the first aspect of the invention maycomprise the probiotic microorganism in any concentration, preferably ata concentration of from 10³ to 10¹⁵ viable cells per gram of the totalcomposition. This concentration of cells provides a suitableconcentration for successful colonization of the gastrointestinal tractand providing the optimum health benefits to the animal. An additionalprobiotic strain may be present at a concentration of from 10³ to 10¹⁵viable cells per gram of the total composition.

According to a second aspect, the present invention provides a methodfor the prevention or reduction of gastrointestinal Campylobacterinfection in a mammalian animal, the method comprising theadministration of a probiotic microorganism to said animal.

Preferably, the probiotic microorganism is comprised in a composition,for example as described above in relation to the first aspect of theinvention.

All preferred features of the first aspect of the invention, also applyto the second.

In the method of the second aspect of the invention, the animal ispreferably in need of the prevention or reduction of gastrointestinalCampylobacter infection.

The administration of the probiotic microorganism may be by any means orpreferably the administration is oral administration (i.e. ingestion).

A third aspect of the present invention provides a probioticmicroorganism for use in preventing or reducing gastrointestinalCampylobacter infection in a mammalian animal.

All preferred features of the first and second aspect of the invention,also apply to the third.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is described with reference to the figures.

FIG. 1: Fecal bacteria counts by Fluorescent in-situ hybridization(FISH): Campylobacter as a % of total population. Showingpost-antibiotic (baseline) levels compared to effect of probiotic+/−supplementation for 10 days or 23 days.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described with reference to thefollowing non-limiting examples:

EXAMPLE 1

Animal Details and Husbandry Conditions

Cats (n=48) housed in catcare 6 were selected for the study (table 1).Catcare 6 had recently been diagnosed with a clinical naturally acquiredCampylobacter infection. The cats were group housed at all times and hadconstant access to fresh water.

Four rooms were selected to undergo probiotic+/− treatment.

In the 10 days prior to the beginning of the probiotic trial, all catswere treated with antibiotics to control the clinical Campylobacterinfection. All cats received Ceporex (1 tablet twice daily for 10 days).Ceporex contains 50 mg cephalexin, a 3^(rd) generation cephalosporinantibiotic.

Feeding Regimen

All cats were group fed according to a standard protocol. Large trays offood containing 400 g/cat, being offered once daily at 2 pm and leftdown overnight. The diet was standard canned Whiskas Beef (chunk inloaf).

Probiotic Dosing Regimen

Cats in the probiotic+ treatment groups (rooms 1 and 2) were orallydosed with 10 mg (1×10⁹ cells) of a freeze-dried preparation ofLactobacillus acidophillus. Deposited under Accession No. NCIMB 41117once daily after feeding, administered in a gelatin capsule. Theprobiotic− groups (rooms 11 and 12) received no capsule.

Dosing commenced immediately after the cessation of antibiotic therapyand continued for 27 days.

Study Design

The study was designed to incorporate measures at key points during theprocess of antibiotic treatment and recovery. The measures taken were:

-   -   Group daily food intakes.    -   Weekly bodyweight.    -   Group feces quality.    -   Bacterial counts by agar culture and FISH.    -   Bacterial profiling by API biochemical fingerprinting and        ribotyping.        Methodology        Food Intakes

Daily food consumption was monitored for each room, being the amountoffered minus that refused. Individual food intakes are not availablefor this study.

Feces Quality

Group feces quality was assessed daily using the Waltham Feces ScoringGuidelines™. Each defecation was graded on a subjective, 17 point scale.Individual feces scores are not available for this study.

Fecal Bacteria Profile

Feces voided overnight were discarded. Every defecation voided between 8am and 4 pm was collected into a clean feces collection pot and used forbacteriological examination. Feces were processed immediately in thelaboratory under appropriate incubation conditions.

The following bacterial groups were quantified using selective agars:

-   -   Anaerobic culture of Lactobacilli on MRSa agar (Oxoid)    -   Micro-aerobic culture of Campylobacter on selective agar (LabM)

In addition, the following bacterial groups were quantified byfluorescence in situ hybridization (FISH):

Clostridia

Lactobacilli

Campylobacter

Methodology for Campylobacter Enumeration Using Selective Agar

A swab of feces was spread onto a plate and incubated micro-aerobically(5% O₂), selecting for single colonies. This method is qualitative anddoes not provide quantitative information.

Statistical Analysis

Data were analyzed using multifactor ANOVA, with antioxidantsupplementation+/− as the second factor and students t test, asappropriate. P<0.05 was considered significant.

Results

Fecal Bacteria

Plate Counts

Lactobacilli were enumerated on three occasions during the study:

towards the end of antibiotic therapy

following 10 days+/− probiotic treatment

following 23 days+/− probiotic treatment

Total Lactobacilli in feces were enumerated using de Man, Rogosa, Sharpe(MRS) agar acidified to a pH of 5.0. There was no significant effect ofprobiotic treatment on absolute numbers of Lactobacilli at any timepoint.

-   -   Campylobacter were enumerated on four occasions during the        study:    -   before the start of antibiotic therapy

towards the end of antibiotic therapy

following 10 days+/− probiotic treatment

following 23 days+/− probiotic treatment TABLE 1 % of feces samples thattested positive for Campylobacter using selective agar. CampylobacterProbiotic+ Probiotic− (log₁₀) % positive n % positive n Pre-antibiotic100  12 100 12 Post antibiotic 50 12  67 12 10 days +/− probiotic 67 12100 11 23 days +/− probiotic 88 17 100 15

This method is qualitative and merely indicates the presence or absenceof Campylobacter in feces samples. Prior to antibiotic therapy, allfeces samples cultured tested positive for Campylobacter, although thiswas decreased to 59% (overall) by antibiotic therapy. Following 10 daysprobiotic+/− supplementation, 100% of feces from the probiotic− grouptested positive for Campylobacter, but this was decreased to 67% in theprobiotic+ group. Following 23 days probiotic+/− supplementation, 100%of feces from the probiotic− group tested positive for Campylobacter,but this was decreased to 88% in the probiotic+ group (Table 1).Probiotic supplementation therefore decreased the prevalence ofCampylobacter positive feces. Re-infection rates were also reduced inthe probiotic+ group with 67% of fecal samples testing positive forCampylobacter ten days post treatment, compared to 100% of feces fromthe probiotic− group. These findings indicate strength resistance ofhealthy cats to infection with Campylobacter species followingsupplementation with Lactobacilli acidophilus (Accession No. NCIMB41117).

Fluorescence in Situ Hybridization

Enumeration of Clostridia, Lactobacilli and Campylobacter by FISH wasconducted on four occasions during the study:

before the start of antibiotic therapy

towards the end of antibiotic therapy

following 10 days+/− probiotic treatment

following 23 days+/− probiotic treatment

Bacterial counts (% total population) are given in Table 2 forCampylobacter and shown graphically in FIG. 1.

There was no significant effect of probiotic supplementation onLactobacilli as a % of the total population or absolute numbers (log₁₀)at any time during the study.

There was a significant difference between probiotic+/− groups inClostridia (as a % of the total population as well as a small (less thanone log₁₀) but significant (p=0.007) difference in absolute numbers)prior to the beginning of antibiotic therapy. This difference betweengroups was, however, eliminated by the antibiotic therapy such that atbaseline both groups were similar. Administration of probioticssignificantly decreased Clostridia (as % of total population) at both 10and 23 days. This decrease was not reflected in absolute numbers ofClostridia, although at 23 days there was a small (less than one log₁₀)although significant (p=0.006) difference between the probiotic+/−groups.

There was no difference in Campylobacter between the groups at baseline.At 10 days+/− probiotic supplementation, Campylobacter (as % totalpopulation) had increased in all 4 groups (FIG. 1). However,Campylobacter (as % of total population) was significantly reduced inprobiotic treated animals compared to negative controls at 10 days(table 2, FIG. 1). Following 23 days probiotic supplementationCampylobacter (as % total population) was decreased compared tobaseline, but was increased compared to baseline in those animals thatdid not receive probiotics. At 23 days Campylobacter (as % of totalpopulation) was significantly lower in probiotic treated animalscompared to negative controls (table 2, FIG. 1). This was reflected inabsolute numbers at 23 days, with a small (less than one log₁₀) butsignificant difference between groups. TABLE 2 Fecal bacteria counts byFISH: Campylobacter as a % of total population. Probiotic+ Probiotic−Significance of Campylobacter mean SD n mean SD n differencePre-antibiotics 14.27 4.92 11 14.48 4.15 10 0.727 Post-antibiotics 6.143.83 10 5.25 2.3 12 0.494 10 days treatment 12.2 4.2 12 19.7 9.2 11 0.0223 days treatment 3.94 2.58 17 14.06 10.0 11 0.001

Probiotic supplementation resulted in little difference in Lactobacillicompared to control animals, as measured by both plate and FISHmethodology. This finding is unusual in relation to previous findings,when probiotics have been shown to increase the number of beneficialLactobacilli, and may be due to the compromised health status of thecats in the current study. These cats all had a clinical infection ofCampylobacter prior to the beginning of the trial and this would beexpected to adversely affect the normal microflora of all cats.

As can be seen, antibiotics decreased the Campylobacter (as a percentageof the total population of fecal bacteria) from 14.38 to 5.69% (P=<0.05,paired T test). At two weeks, Campylobacter levels had risen in bothgroups, however, the rise in the probiotic+ group was significantly lessthan in the probiotic− group (12.2 and 19.7% of total population,respectively, P=<0.05). Although the organism count decreased in bothgroups at four weeks, elimination from the probiotic+ group cats wasmarkedly accelerated (14.06% of total population in probiotic− and 3.94%of total population in probiotic+ cats, P=<0.05).

Probiotic supplementation significantly decreased the levels ofpotentially pathogenic Campylobacter compared to cats that had receivedno probiotics.

The study described herein demonstrates that Lactobacillus acidophiluscan improve recovery of the feline gastrointestinal tract from theeffects of antibiotic therapy, by decreasing the number of Campylobacteras a % of the total population. This would be expected to decreaserecovery time of the cat and therefore decrease the zoonotic risk fromfecal shedding of Campylobacter.

EXAMPLE 2

Determination of the Anti-Campylobacter Activity of ProbioticMicroorganism

Objective

In this study, the ability of potential probiotic strains of bacteria tohave an antibacterial effect on Campylobacter jejuni is addressed.

Materials and Methods

Bacterial Strains and Culture Conditions

Campylobacter jejuni cultures were maintained on Mueller Hinton agar(Oxoid) and used as an inoculum for liquid cultures (Mueller Hintonbroth, Oxoid) that were grown in 20 ml volumes in 50 ml conical flasksshaken on an orbital shaker.

Potential probiotic strains were maintained on MRS agar and cultured in20 ml volumes in MRS broth under anaerobic conditions.

Experimental Set-Up

-   (i) Liquid cultures of probiotic strains were set up and incubated    overnight under appropriate conditions. A 1 μl loopful of the    overnight culture was then used to inoculate the very centre of a    150 mm MRS agar plate. These large plates were incubated    anaerobically overnight to allow the growth from the spot inoculum.-   (ii) Pathogenic liquid cultures were set up on the same day as the    probiotic spot cultures and incubated overnight. Overnight pathogen    cultures were adjusted to A₆₀₀ 0.4 before inclusion in the assay.-   (iii) To 15 ml of molten MH agar, 200 μl of the adjusted pathogen    culture was added and swirled gently to mix. This agar/pathogen mix    was then poured into a 90 mm petri dish and allowed to set.-   (iv) When pathogen inoculated agar set it was aseptically removed    from the petri dish. Two sterile disposable loops were used to    remove the agar by gently lifting it away from the dish and slowly    lowering the agar disc onto the spot of probiotic growth on the 150    mm agar plates.-   (v) The agar “sandwich” was incubated overnight at 37° C. under    aerobic conditions.-   (vi) After overnight incubation, the zone of no bacterial growth    over the probiotic spot was measured and the diameter of the    probiotic spot subtracted from this figure. The resulting value is    taken as the zone of inhibition.-   (vii) All experiments were carried out a minimum of three times for    each strain-pathogen combination.    Results    Anti-Campylobacter Potential of Probiotic Strains

Following incubation of the potential probiotic strains withCampylobacter jejuni the zones of inhibition were determined for eachstrain (see Table 3 below). TABLE 3 Probiotic Strain Average InhibitionZone (mm) L. acidophilus 19.3 L. ruminus 16.3 L. reuteri  5.3 L. murinus 9.3 L. mucosae  2.7 L. casei 21.3Discussion

The anti-Campylobacter activity of the strains is clearly demonstrated.

1. A method of manufacturing a composition for the prevention orreduction of gastrointestinal Campylobacter infection, in a mammaliananimal, said method comprises the step of adding a probioticmicroorganism to the composition.
 2. The method as claimed in claim 1,wherein the probiotic microorganism is Lactobacillus.
 3. The method, asclaimed in claim 2, wherein the probiotic microorganism is Lactobacillusacidophilus.
 4. The method, as claimed in claim 1, wherein theCampylobacter is Campylobacter jejuni.
 5. The method as claimed in claim1, wherein the mammalian animal is a dog, cat or a human.
 6. The method,as claimed in claim 1, wherein the composition is a foodstuff.
 7. Themethod, as claimed in claim 6, wherein the foodstuff is a dry pet food.8. A method for the prevention or reduction of gastrointestinalCampylobacter infection in a mammalian animal, the method comprising theadministration of a probiotic microorganism to said animal.
 9. Themethod, as claimed in claim 8, wherein the probiotic microorganism iscomprised in a composition.
 10. The method as claimed in claim 9,wherein the composition is a foodstuff.
 11. The method, as claimed inclaim 10, wherein the foodstuff is a dry pet food.
 12. The method, asclaimed in claim 8, wherein the administration is by oral ingestion. 13.The method, as claimed in claim 8, wherein the probiotic microorganismis Lactobacillus.
 14. The method, as claimed in claim 13, wherein theprobiotic microorganism is Lactobacillus acidophilus.
 15. The method, asclaimed claim 8, wherein the Campylobacter infection is Campylobacterjejuni.
 16. The method, as claimed in claim 8, wherein the animal is acat, dog or a human.
 17. A probiotic microorganism, for use inpreventing or reducing gastrointestinal Campylobacter infection in amammalian animal.
 18. The probiotic microorganism, as claimed in claim17, which is comprised in a composition.
 19. (canceled)
 20. (canceled)